Sodium taurocholate cotransporting polypeptide (NTCP) involved in bile acid transport in the liver is an entry receptor of hepatitis B virus (HBV). In the present study, we introduce a mass spectrometric screening assay for targeting HBV entry inhibitors that can reduce NTCP transporter activity by employing taurocholic acid (TCA) labeled with stable isotope (2,2,4,4-d4-TCA, d4-TCA) and NTCP-overexpressing human liver cancer cell lines such as HepG2 and Huh-7. The accuracy and reliability of the proposed mass spectrometric NTCP activity assay have been validated with known HBV inhibitors including cyclosporine A (CsA) and pre-S1 peptide (PreS/2-48Myr or myrcludex B analog) that suppress the entry of HBV into hepatocytes by targeting NTCP. For the inhibitor screening assay, NTCP-overexpressing HepG2 or Huh-7 cells are treated with either a combination of TCA and an inhibitor (CsA or PreS/2-48Myr) or d4-TCA alone to serve as a reference. The activity of an HBV inhibitor is determined by relative quantification between TCA and d4-TCA in a 1:1 mixture of inhibitor-treated cells and untreated control cells using liquid chromatography-mass spectrometry. With our new approach, the half maximal inhibitory concentration (IC50) values for CsA and PreS/2-48Myr have been determined at micromolar and nanomolar concentrations, respectively, which is consistent with the previous results obtained with other conventional HBV entry inhibitor assay methods. Our assay method does not require HBV infection or radioactive 3H-TCA and provides a facile way to identify viral entry inhibitors via measuring bile acid transport activity of NTCP.